Chang-Ro Lee

Global Dissemination of Carbapenemase-Producing Klebsiella pneumoniae: Epidemiology, Genetic Context, Treatment Options, and Detection Methods

The emergence of carbapenem-resistant Gram-negative pathogens poses a serious threat to public health worldwide. In particular, the increasing prevalence of carbapenem-resistant Klebsiella pneumoniae is a major source of concern. K. pneumoniae carbapenemases (KPCs) and carbapenemases of the oxacillinase-48 (OXA-48) type have been reported worldwide. New Delhi metallo-β-lactamase (NDM) carbapenemases were originally identified in Sweden in 2008 and have spread worldwide rapidly. In this review, we summarize the epidemiology of K. pneumoniae producing three carbapenemases (KPCs, NDMs, and OXA-48-like). Although the prevalence of each resistant strain varies geographically, K. pneumoniae producing KPCs, NDMs, and OXA-48-like carbapenemases have become rapidly disseminated. In addition, we used recently published molecular and genetic studies to analyze the mechanisms by which these three carbapenemases, and major K. pneumoniae clones, such as ST258 and ST11, have become globally prevalent. Because carbapenemase-producing K. pneumoniae are often resistant to most β-lactam antibiotics and many other non-β-lactam molecules, the therapeutic options available to treat infection with these strains are limited to colistin, polymyxin B, fosfomycin, tigecycline, and selected aminoglycosides. Although, combination therapy has been recommended for the treatment of severe carbapenemase-producing K. pneumoniae infections, the clinical evidence for this strategy is currently limited, and more accurate randomized controlled trials will be required to establish the most effective treatment regimen. Moreover, because rapid and accurate identification of the carbapenemase type found in K. pneumoniae may be difficult to achieve through phenotypic antibiotic susceptibility tests, novel molecular detection techniques are currently being developed.

Escherichia coli enzyme IIANtr regulates the K+ transporter TrkA

The maintenance of ionic homeostasis in response to changes in the environment is essential for all living cells. Although there are still many important questions concerning the role of the major monovalent cation K+, cytoplasmic K+ in bacteria is required for diverse processes. Here, we show that enzyme IIANtr (EIIANtr) of the nitrogen-metabolic phosphotransferase system interacts with and regulates the Escherichia coli K+ transporter TrkA. Previously we reported that an E. coli K-12 mutant in the ptsN gene encoding EIIANtr was extremely sensitive to growth inhibition by leucine or leucine-containing peptides (LCPs). This sensitivity was due to the requirement of the dephosphorylated form of EIIANtr for the derepression of ilvBN expression. Whereas the ptsN mutant is extremely sensitive to LCPs, a ptsN trkA double mutant is as resistant as WT. Furthermore, the sensitivity of the ptsN mutant to LCPs decreases as the K+ level in culture media is lowered. We demonstrate that dephosphorylated EIIANtr, but not its phosphorylated form, forms a tight complex with TrkA that inhibits the accumulation of high intracellular concentrations of K+. High cellular K+ levels in a ptsN mutant promote the sensitivity of E. coli K-12 to leucine or LCPs by inhibiting both the expression of ilvBN and the activity of its gene products. Here, we delineate the similarity of regulatory mechanisms for the paralogous carbon and nitrogen phosphotransferase systems. Dephosphorylated EIIAGlc regulates a variety of transport systems for carbon sources, whereas dephosphorylated EIIANtr regulates the transport system for K+, which has global effects related to nitrogen metabolism.

Strategies to Minimize Antibiotic Resistance

Antibiotic resistance can be reduced by using antibiotics prudently based on guidelines of antimicrobial stewardship programs (ASPs) and various data such as pharmacokinetic (PK) and pharmacodynamic (PD) properties of antibiotics, diagnostic testing, antimicrobial susceptibility testing (AST), clinical response, and effects on the microbiota, as well as by new antibiotic developments. The controlled use of antibiotics in food animals is another cornerstone among efforts to reduce antibiotic resistance. All major resistance-control strategies recommend education for patients, children (e.g., through schools and day care), the public, and relevant healthcare professionals (e.g., primary-care physicians, pharmacists, and medical students) regarding unique features of bacterial infections and antibiotics, prudent antibiotic prescribing as a positive construct, and personal hygiene (e.g., handwashing). The problem of antibiotic resistance can be minimized only by concerted efforts of all members of society for ensuring the continued efficiency of antibiotics.

Selective fluorescent chemosensor for the bacterial alarmone (p)ppGpp

We have developed the first selective fluorescent chemosensor (PyDPA) for (p)ppGpp, a bacterial and plant alarmone. By using pyrene-excimer fluorescence, PyDPA shows very good selectivity for (p)ppGpp from among other nucleotides in water. PyDPA was used for the real-time detection of in vitro ppGpp synthesis by bacterial ribosomal complexes.

Requirement of the dephospho-form of enzyme IIANtr for derepression of Escherichia coli K-12 ilvBN expression.

While the proteins of the phosphoenolpyruvate:carbohydrate phosphotransferase system (carbohydrate PTS) have been shown to regulate numerous targets, little such information is available for the nitrogen‐metabolic phosphotransferase system (nitrogen‐metabolic PTS). To elucidate the physiological role of the nitrogen‐metabolic PTS, we carried out phenotype microarray (PM) analysis with Escherichia coli K‐12 strain MG1655 deleted for the ptsP gene encoding the first enzyme of the nitrogen‐metabolic PTS. Together with the PM data, growth studies revealed that a ptsN (encoding enzyme IIANtr) mutant became extremely sensitive to leucine‐containing peptides (LCPs), while both ptsP (encoding enzyme INtr) and ptsO (encoding NPr) mutants were more resistant than wild type. The toxicity of LCPs was found to be due to leucine and the dephospho‐form of enzyme IIANtr was found to be necessary to neutralize leucine toxicity. Further studies showed that the dephospho‐form of enzyme IIANtr is required for derepression of the ilvBN operon encoding acetohydroxy acid synthase I catalysing the first step common to the biosynthesis of the branched‐chain amino acids.

Salmonella pathogenicity island 2 expression negatively controlled by EIIANtr–SsrB interaction is required for Salmonella virulence

SsrA/SsrB is a primary two-component system that mediates the survival and replication of Salmonella within host cells. When activated, the SsrB response regulator directly promotes the transcription of multiple genes within Salmonella pathogenicity island 2 (SPI-2). As expression of the SsrB protein is promoted by several transcription factors, including SsrB itself, the expression of SPI-2 genes can increase to undesirable levels under activating conditions. Here, we report that Salmonella can avoid the hyperactivation of SPI-2 genes by using ptsN-encoded EIIANtr, a component of the nitrogen-metabolic phosphotransferase system. Under SPI-2–inducing conditions, the levels of SsrB-regulated gene transcription increased abnormally in a ptsN deletion mutant, whereas they decreased in a strain overexpressing EIIANtr. We found that EIIANtr controls SPI-2 genes by acting on the SsrB protein at the posttranscriptional level. EIIANtr interacted directly with SsrB, which prevented the SsrB protein from binding to its target promoter. Finally, the Salmonella strain, either lacking the ptsN gene or overexpressing EIIANtr, was unable to replicate within macrophages, and the ptsN deletion mutant was attenuated for virulence in mice. These results indicated that normal SPI-2 gene expression maintained by an EIIANtr–SsrB interaction is another determinant of Salmonella virulence.

Reciprocal regulation of the autophosphorylation of enzyme INtr by glutamine and α-ketoglutarate in Escherichia coli

In addition to the phosphoenolpyruvate:sugar phosphotransferase system (sugar PTS), most proteobacteria possess a paralogous system (nitrogen phosphotransferase system, PTSNtr). The first proteins in both pathways are enzymes (enzyme Isugar and enzyme INtr) that can be autophosphorylated by phosphoenolpyruvate. The most striking difference between enzyme Isugar and enzyme INtr is the presence of a GAF domain at the N‐terminus of enzyme INtr. Since the PTSNtr was identified in 1995, it has been implicated in a variety of cellular processes in many proteobacteria and many of these regulations have been shown to be dependent on the phosphorylation state of PTSNtr components. However, there has been little evidence that any component of this so‐called PTSNtr is directly involved in nitrogen metabolism. Moreover, a signal regulating the phosphorylation state of the PTSNtr had not been uncovered. Here, we demonstrate that glutamine and α‐ketoglutarate, the canonical signals of nitrogen availability, reciprocally regulate the phosphorylation state of the PTSNtr by direct effects on enzyme INtr autophosphorylation and the GAF signal transduction domain is necessary for the regulation of enzyme INtr activity by the two signal molecules. Taken together, our results suggest that the PTSNtr senses nitrogen availability.

Potassium Mediates Escherichia coli Enzyme IIA Ntr -dependent Regulation of Sigma Factor Selectivity

An Escherichia coli mutant devoid of enzyme IIANtr (EIIANtr) of the nitrogen PTS is extremely sensitive to leucine‐containing peptides due to decreased expression of acetohydroxy acid synthase. This decreased expression is due to defective potassium homeostasis. We further elucidate here the mechanism for regulation of gene expression by the intracellular level of K+. The leucine hypersensitivity of a ptsN (encoding EIIANtr) mutant was suppressed by deleting rpoS, encoding the stationary phase σ factor. Despite intracellular levels of sigma factors comparable to the wild‐type strain, most of the genes downregulated in a ptsN mutant are controlled by σ70, while all the upregulated genes are controlled by σS, implying that the balance of sigma activities is modified by ptsN deletion. This change of sigma factor activities in the deletion mutant was found to be due to increased levels of K+. In vitro transcription assays demonstrated that a σ70 controlled gene and a σS controlled gene were differentially affected by potassium concentration. Biochemical studies revealed that K+ is responsible for sigma factor competition by differentially influencing the binding of σ70 and σS to core RNA polymerase. Taken together, the data suggest that EIIANtr controls sigma factor selectivity by regulating the intracellular K+ level.

Biology of Acinetobacter baumannii: Pathogenesis, Antibiotic Resistance Mechanisms, and Prospective Treatment Options

Acinetobacter baumannii is undoubtedly one of the most successful pathogens responsible for hospital-acquired nosocomial infections in the modern healthcare system. Due to the prevalence of infections and outbreaks caused by multi-drug resistant A. baumannii, few antibiotics are effective for treating infections caused by this pathogen. To overcome this problem, knowledge of the pathogenesis and antibiotic resistance mechanisms of A. baumannii is important. In this review, we summarize current studies on the virulence factors that contribute to A. baumannii pathogenesis, including porins, capsular polysaccharides, lipopolysaccharides, phospholipases, outer membrane vesicles, metal acquisition systems, and protein secretion systems. Mechanisms of antibiotic resistance of this organism, including acquirement of β-lactamases, up-regulation of multidrug efflux pumps, modification of aminoglycosides, permeability defects, and alteration of target sites, are also discussed. Lastly, novel prospective treatment options for infections caused by multi-drug resistant A. baumannii are summarized.

Dephosphorylated NPr of the nitrogen PTS regulates lipid A biosynthesis by direct interaction with LpxD.

Bacterial phosphoenolpyruvate-dependent phosphotransferase systems (PTS) play multiple roles in addition to sugar transport. Recent studies revealed that enzyme IIA(Ntr) of the nitrogen PTS regulates the intracellular concentration of K(+) by direct interaction with TrkA and KdpD. In this study, we show that dephosphorylated NPr of the nitrogen PTS interacts with Escherichia coli LpxD which catalyzes biosynthesis of lipid A of the lipopolysaccharide (LPS) layer. Mutations in lipid A biosynthetic genes such as lpxD are known to confer hypersensitivity to hydrophobic antibiotics such as rifampin; a ptsO (encoding NPr) deletion mutant showed increased resistance to rifampin and increased LPS biosynthesis. Taken together, our data suggest that unphosphorylated NPr decreases lipid A biosynthesis by inhibiting LpxD activity.